WO2016117567A1 - Separation material - Google Patents
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- WO2016117567A1 WO2016117567A1 PCT/JP2016/051460 JP2016051460W WO2016117567A1 WO 2016117567 A1 WO2016117567 A1 WO 2016117567A1 JP 2016051460 W JP2016051460 W JP 2016051460W WO 2016117567 A1 WO2016117567 A1 WO 2016117567A1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28047—Gels
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
- B01J20/28057—Surface area, e.g. B.E.T specific surface area
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
- B01J20/28095—Shape or type of pores, voids, channels, ducts
- B01J20/28097—Shape or type of pores, voids, channels, ducts being coated, filled or plugged with specific compounds
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
- B01J20/3206—Organic carriers, supports or substrates
- B01J20/3208—Polymeric carriers, supports or substrates
- B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
- B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/328—Polymers on the carrier being further modified
- B01J20/3282—Crosslinked polymers
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J41/00—Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
- B01J41/20—Anion exchangers for chromatographic processes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
Definitions
- the present invention relates to a separating material.
- ion exchangers based on porous synthetic polymers, particles based on cross-linked gels of hydrophilic natural polymers, etc. It is used.
- the ion exchanger based on the above porous synthetic polymer has an advantage that the pressure resistance during liquid passage is good.
- nonspecific adsorption such as irreversible adsorption based on hydrophobic interaction occurs, resulting in peak asymmetry, or ion exchange by hydrophobic interaction.
- the protein adsorbed on the body may not be recovered while adsorbed.
- Patent Document 1 describes that by using such a complex, the load factor of the reactive substance is increased, and high yield synthesis is possible.
- Patent Document 2 An ion exchanger of a hybrid copolymer in which pores of a so-called macro network structure copolymer are filled with a crosslinked copolymer gel synthesized from a monomer is known (see Patent Document 2).
- Crosslinked copolymer gels have problems such as pressure loss and volume change when the degree of crosslinking is low.
- Patent Document 2 describes that the leakage behavior is improved.
- Patent Document 5 A technique for synthesizing porous particles composed of glycidyl methacrylate and an acrylic crosslinked monomer is known (see Patent Document 5).
- an object of the present invention is to provide a separation material that ensures liquid permeability and can reduce non-specific adsorption of proteins, increase the amount of adsorption, and suppress denaturation.
- the present invention provides the separating material described in [1] to [6] below.
- the polymer unit has at least one of styrene and divinylbenzene at 90% by mass or more based on the total amount of the monomer, and has a hydroxyl group that covers at least a part of the surface of the porous polymer particle.
- a coating layer containing a polymer wherein the coating amount by the coating layer is 1 to 15 mg / m 2 per unit specific surface area of the porous polymer particles.
- the separation material according to [1] wherein the oxygen element ratio on the surface of the separation material is 10 to 50%.
- the present invention it is possible to provide a separation material that ensures liquid permeability and can reduce non-specific adsorption of proteins, increase the amount of adsorption, and suppress denaturation.
- the separating material of the present embodiment includes porous polymer particles and a coating layer that covers at least part of the surface of the porous polymer particles.
- the “surface of the porous polymer particle” includes not only the outer surface of the porous polymer particle but also the surface of the pores inside the porous polymer particle.
- the porous polymer particles of the present embodiment are particles obtained by curing a monomer containing a porosifying agent, and can be synthesized by, for example, conventional suspension polymerization or emulsion polymerization.
- the monomer unit at least one of styrene and divinylbenzene contains 90% by mass or more based on the total monomer amount, and preferably 90% by mass or more of divinylbenzene based on the total monomer amount. By containing a predetermined amount of styrene or divinylbenzene, the pressure resistance tends to be excellent.
- the monomer may further contain the following polyfunctional monomers other than styrene and divinylbenzene, monofunctional monomers, and the like.
- polyfunctional monomers other than divinylbenzene examples include divinyl compounds such as divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene. These polyfunctional monomers can be used alone or in combination of two or more.
- Monofunctional monomers other than styrene include, for example, o-methylstyrene, m-methylstyrene, p-methylstyrene, ⁇ -methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2,4 -Dimethylstyrene, pn-butylstyrene, pt-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn- Examples thereof include styrene derivatives such as dodecylstyrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene,
- styrene derivative which has functional groups, such as a carboxy group, an amino group, a hydroxyl group, and an aldehyde group, can also be used.
- the porosifying agent examples include aliphatic or aromatic hydrocarbons, esters, ketones, ethers, alcohols, and the like, which are organic solvents that promote phase separation at the time of polymerization and promote pore formation of particles. It is done. Specific examples include toluene, xylene, diethylbenzene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol, cyclohexanol and the like. These porous agents can be used singly or in combination of two or more.
- the above porosifying agent can be used in an amount of 0 to 200% by mass based on the total mass of the monomer.
- the porosity of the porous polymer particles can be controlled by the amount of the porous agent.
- the size and shape of the pores of the porous polymer particles can be controlled by the kind of the porous agent.
- Water used as a solvent can be used as a porous agent.
- water is used as a porosifying agent, it is possible to make it porous by dissolving an oil-soluble surfactant in the monomer and absorbing water.
- oil-soluble surfactant used for the porosification examples include sorbitan monoesters of branched C16 to C24 fatty acids, chain unsaturated C16 to C22 fatty acids or chain saturated C12 to C14 fatty acids, such as sorbitan monolaurate, sorbitan Sorbitan monoesters derived from monooleate, sorbitan monomyristate or coconut fatty acid; diglycerol monoesters of branched C16-C24 fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12-C14 fatty acids, for example di- Glycerol monooleate (for example, diglycerol monoester of C18: 1 (18 carbon atoms, 1 double bond) fatty acid), diglycerol monomyristate, diglycerol monoisostearate or diglycerol monoester of coconut fatty acid Ester; Branch C16 ⁇ 24 alcohol (e.g., Guerbet alcohols), linear unsaturated C16 ⁇ C24
- sorbitan monolaurate e.g., SPAN 20
- Sorbitan monooleate e.g, SPAN 80, preferably about 40% purity, more preferably about 50% purity, most preferably greater than about 70% purity sorbitan monooleate
- Glycerol monooleate eg, greater than about 40% purity, more preferably greater than about 50% purity, most preferably greater than about 70% purity
- diglycerol monoisostearate eg, preferably Is greater than about 40% purity, more preferably greater than about 50% purity
- diglycerol monomyristate preferably greater than about 40% purity, more preferably greater than about 50% purity, most preferably about 70% purity.
- cocoyl eg, lauryl, myristoyl, etc.
- oil-soluble surfactants are preferably used in the range of 5 to 80% by mass relative to the total mass of the monomer. If the content of the oil-soluble surfactant is 5% by mass or more, the stability of the water droplet is sufficient, and it is difficult to form a large single hole. Further, when the content of the oil-soluble surfactant is 80% by mass or less, it becomes easier for the porous polymer particles to retain the shape after polymerization.
- aqueous medium used for the polymerization reaction examples include water, a mixed medium of water and a water-soluble solvent (for example, lower alcohol), and the like.
- the aqueous medium may contain a surfactant.
- the surfactant any of anionic, cationic, nonionic and zwitterionic surfactants can be used.
- anionic surfactant examples include fatty acid oils such as sodium oleate and castor oil potassium, alkyl sulfate salts such as sodium lauryl sulfate and ammonium lauryl sulfate, alkylbenzene sulfonates such as sodium dodecylbenzenesulfonate, and alkylnaphthalene sulfone.
- Acid salts alkane sulfonates, dialkyl sulfosuccinates such as sodium dioctyl sulfosuccinate, alkenyl succinates (dipotassium salts), alkyl phosphate esters, naphthalene sulfonate formalin condensates, polyoxyethylene alkylphenyl ether sulfates Salts, polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylene lauryl ether sulfate, polyoxyethylene alkyl sulfates, etc.
- cationic surfactant examples include alkylamine salts such as laurylamine acetate and stearylamine acetate, and quaternary ammonium salts such as lauryltrimethylammonium chloride.
- Nonionic surfactants include, for example, hydrocarbon nonionic surfactants such as polyethylene glycol alkyl ethers, polyethylene glycol alkyl aryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, polyalkylene glycol alkylamines, or amides.
- hydrocarbon nonionic surfactants such as polyethylene glycol alkyl ethers, polyethylene glycol alkyl aryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, polyalkylene glycol alkylamines, or amides.
- Agents polyether-modified silicon-based nonionic surfactants such as polyethylene oxide adducts of silicon and polypropylene oxide adducts, and fluorine-based nonionic surfactants such as perfluoroalkyl glycols.
- zwitterionic surfactants include hydrocarbon surfactants such as lauryl dimethylamine oxide, phosphate ester surfactants, and phosphite ester surfactants.
- Surfactant may be used alone or in combination of two or more.
- anionic surfactants are preferable from the viewpoint of dispersion stability during monomer polymerization.
- polymerization initiator examples include benzoyl peroxide, lauroyl peroxide, orthochlorobenzoyl peroxide, orthomethoxybenzoyl peroxide, 3,5,5-trimethylhexanoyl peroxide, tert-butyl peroxide.
- Organic peroxides such as oxy-2-ethylhexanoate and di-tert-butyl peroxide; 2,2′-azobisisobutyronitrile, 1,1′-azobiscyclohexanecarbonitrile, 2,2 ′ -Azo compounds such as azobis (2,4-dimethylvaleronitrile).
- the polymerization initiator can be used in the range of 0.1 to 7.0 parts by mass with respect to 100 parts by mass of the monomer.
- the polymerization temperature can be appropriately selected according to the type of monomer and polymerization initiator.
- the polymerization temperature is preferably 25 to 110 ° C, more preferably 50 to 100 ° C.
- a polymer dispersion stabilizer may be used in order to improve the dispersion stability of the particles.
- polymer dispersion stabilizer examples include inorganic water-soluble high polymers such as polyvinyl alcohol, polycarboxylic acid, celluloses (hydroxyethyl cellulose, carboxymethyl cellulose, methyl cellulose, etc.), polyvinyl pyrrolidone, tricalcium phosphate (TCP), sodium tripolyphosphate, and the like. Examples include molecular compounds. These can be used individually or in combination of multiple types. Of these, tricalcium phosphate (TCP), polyvinyl alcohol, or polyvinylpyrrolidone is preferred.
- the addition amount of the polymer dispersion stabilizer is preferably 1 to 10 parts by mass with respect to 100 parts by mass of the monomer.
- a water-soluble polymerization inhibitor such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble vitamin Bs, citric acid, polyphenols and the like may be used.
- the average particle diameter of the porous polymer particles is preferably 300 ⁇ m or less, more preferably 150 ⁇ m or less, and even more preferably 100 ⁇ m or less. Further, the average particle diameter of the porous polymer particles is preferably 10 ⁇ m or more, more preferably 30 ⁇ m or more, and further preferably 50 ⁇ m or more, from the viewpoint of improving liquid permeability.
- the coefficient of variation (CV) of the particle size of the porous polymer particles is preferably 3 to 15%, more preferably 5 to 15%, from the viewpoint of improving liquid permeability. More preferably, it is 10%.
- CV coefficient of variation
- C. of average particle size and particle size of porous polymer particles or separator V. can be determined by the following measurement method. 1) Disperse the particles in water (including a dispersant such as a surfactant) using an ultrasonic dispersion device to prepare a dispersion liquid containing 1% by mass of porous polymer particles. 2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex Corporation), an average particle size and particle size of C.I. V. (Coefficient of variation) is measured.
- the pore volume (porosity) of the porous polymer particles is preferably 30% by volume or more and 70% by volume or less, and 40% by volume or more and 70% by volume based on the total volume (including the pore volume) of the porous polymer particles. % Or less is more preferable.
- the porous polymer particles preferably have pores having a mode diameter (mode value of pore diameter distribution, maximum frequency pore diameter, average pore diameter) of 0.05 to 1 ⁇ m in the pore diameter distribution.
- the mode diameter in the pore size distribution of the porous polymer particles is more preferably 0.2 ⁇ m or more and less than 0.5 ⁇ m. When the mode diameter in the pore size distribution is 0.05 ⁇ m or more, a substance tends to easily enter the pores. When the mode diameter in the pore diameter distribution is 1 ⁇ m or less, the specific surface area is sufficient. These can be adjusted by the above-mentioned porous agent.
- the specific surface area of the porous polymer particles is preferably 10 m 2 / g or more, and more preferably 30 m 2 / g or more. From the viewpoint of higher practicality, the specific surface area is more preferably 35 m 2 / g or more, and further preferably 40 m 2 / g or more. When the specific surface area is 10 m 2 / g or more, the adsorption amount of the substance to be separated tends to increase.
- the upper limit of the specific surface area of the porous polymer particles is not particularly limited, but can be, for example, 200 m 2 / g or less, 100 m 2 / g or less.
- the coating layer of this embodiment contains a polymer having a predetermined amount (coating amount) of hydroxyl groups.
- a polymer having a predetermined amount of hydroxyl groups By covering the porous polymer particles with a polymer having a predetermined amount of hydroxyl groups, the increase in column pressure can be suppressed, and nonspecific adsorption of proteins can be suppressed, and the protein adsorption of the separation material The amount tends to be good. Furthermore, when the polymer having a hydroxyl group is crosslinked, it is possible to further suppress an increase in column pressure.
- the coating amount by the coating layer is 1 to 15 mg / m 2 per unit specific surface area of the porous polymer particles, preferably 5 to 15 mg / m 2 , and preferably 8 to 15 mg / m 2. 2 is more preferable.
- the coating amount by the coating layer can be measured by the method described in the examples.
- the oxygen element ratio on the surface is preferably 10 to 50%, more preferably 15 to 50%, further preferably 20 to 50%, and more preferably 30 to 50%. And particularly preferred.
- the oxygen element ratio on the surface of the separating material can be measured by the method described in the examples.
- the oxygen element ratio on the surface of the separating material is measured by X-ray photoelectron spectroscopy in which the coating layer is irradiated with X-rays from the outside of the separating material.
- the polymer having a hydroxyl group preferably has two or more hydroxyl groups in one molecule, and more preferably a hydrophilic polymer.
- examples of the polymer having a hydroxyl group include polysaccharides and polyvinyl alcohol.
- Preferred examples of the polysaccharide include agarose, dextran, cellulose, chitosan, and alginic acid.
- a polymer having a weight average molecular weight of about 10,000 to 200,000 can be used.
- the polymer having a hydroxyl group is preferably a modified product modified with a hydrophobic group from the viewpoint of improving the interfacial adsorption ability.
- the hydrophobic group include an alkyl group having 1 to 6 carbon atoms and an aryl group having 6 to 10 carbon atoms.
- the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, and a propyl group.
- Examples of the aryl group having 6 to 10 carbon atoms include a phenyl group and a naphthyl group.
- a hydrophobic group is introduced by reacting a functional group that reacts with a hydroxyl group (for example, an epoxy group) and a compound having a hydrophobic group (for example, glycidyl phenyl ether) with a polymer having a hydroxyl group by a conventionally known method. Can do.
- a functional group that reacts with a hydroxyl group for example, an epoxy group
- a compound having a hydrophobic group for example, glycidyl phenyl ether
- the coating layer containing a polymer having a hydroxyl group can be formed by, for example, the following method.
- a polymer solution having a hydroxyl group is adsorbed on the surface of the porous polymer particles.
- the solvent for the polymer solution having a hydroxyl group is not particularly limited as long as it can dissolve the polymer having a hydroxyl group, but water is the most common.
- the concentration of the polymer dissolved in the solvent is preferably 5 to 20 (mg / mL).
- This solution is impregnated into porous polymer particles.
- porous polymer particles are added to a polymer solution having a hydroxyl group and left for a predetermined time.
- the impregnation time varies depending on the surface state of the porous body
- the polymer concentration is in an equilibrium state with the external concentration inside the porous body if it is usually impregnated day and night. Then, it wash
- Crosslinking treatment Next, a crosslinking agent is added to cause the polymer having a hydroxyl group adsorbed on the surface of the porous polymer particles to undergo a crosslinking reaction to form a crosslinked body. That is, the polymer having a hydroxyl group of the separating material may be cross-linked. At this time, the crosslinked body has a three-dimensional crosslinked network structure having a hydroxyl group.
- an epihalohydrin such as epichlorohydrin
- a dialdehyde compound such as glutaraldehyde
- a diisocyanate compound such as methylene diisocyanate
- a glycidyl compound such as ethylene glycol diglycidyl ether
- two or more functional groups active on a hydroxyl group The compound which has is mentioned.
- a dihalide such as dichlorooctane can also be used as a crosslinking agent.
- a catalyst is usually used for this crosslinking reaction.
- a conventionally known catalyst can be appropriately used according to the type of the crosslinking agent.
- the crosslinking agent is epichlorohydrin or the like
- an alkali such as sodium hydroxide
- mineral acids such as hydrochloric acid are effective.
- the crosslinking reaction with the crosslinking agent is usually performed by adding the crosslinking agent to a system in which the separating material is dispersed and suspended in an appropriate medium.
- the addition amount of the crosslinking agent is, for example, within a range of 0.1 to 100 moles per unit of one unit of monosaccharide. It can be selected according to performance.
- the addition amount of the crosslinking agent is reduced, the coating layer tends to be easily peeled off from the porous polymer particles.
- the addition amount of a crosslinking agent is excessive and the reaction rate with the polymer which has a hydroxyl group is high, the characteristic of the polymer which has a hydroxyl group of a raw material tends to be impaired.
- the amount of the catalyst used varies depending on the type of the crosslinking agent. Usually, when a polysaccharide is used as the polymer having a hydroxyl group, if one unit of the monosaccharide forming the polysaccharide is 1 mole, It is preferably used in the range of 0.01 to 10 mole times, more preferably 0.1 to 5 mole times.
- the cross-linking reaction condition is a temperature condition
- the temperature of the reaction system is raised, and the cross-linking reaction occurs when the temperature reaches the reaction temperature.
- the polymer or crosslinking agent is not extracted from the impregnated polymer solution, and a crosslinking reaction is performed.
- the crosslinking reaction is usually performed at a temperature in the range of 5 to 90 ° C. for 1 to 10 hours.
- the temperature is preferably in the range of 30 to 90 ° C.
- the produced particles are filtered, washed with a hydrophilic organic solvent such as water, methanol, ethanol, etc. to remove unreacted polymer, suspending medium, etc.
- a hydrophilic organic solvent such as water, methanol, ethanol, etc.
- the separation material having a coating layer can be used for ion exchange purification, affinity purification, etc. by introducing ion exchange groups, ligands (protein A), etc. via hydroxyl groups on the surface.
- Examples of the method for introducing an ion exchange group include a method using an alkyl halide compound.
- halogenated alkyl compound examples include monohalogenocarboxylic acids such as monohalogenoacetic acid and monohalogenopropionic acid and sodium salts thereof, primary, secondary or tertiary amines having at least one halogenated alkyl group such as diethylaminoethyl chloride, halogen And quaternary ammonium hydrochloride having an alkyl group.
- halogenated alkyl compounds are preferably bromides or chlorides.
- the amount of the halogenated alkyl compound used is preferably 0.2% by mass or more based on the total mass of the separating material imparting ion exchange groups.
- organic solvent examples include alcohols such as ethanol, 1-propanol, 2-propanol, 1-butanol, isobutanol, 1-pentanol, and isopentanol.
- the ion exchange group is introduced into the hydroxyl group on the surface of the separation material, the wet particles are drained by filtration or the like, immersed in an alkaline aqueous solution of a predetermined concentration, left for a certain time, and then water-organic.
- the halogenated alkyl compound is added and reacted in a solvent mixture system. This reaction is preferably carried out at a temperature of 40 to 90 ° C. for 0.5 to 12 hours.
- the ion exchange group to be provided is determined depending on the kind of the halogenated alkyl compound used in the above reaction.
- a mono- having at least one alkyl group in which a part of hydrogen atoms is substituted with a chlorine atom Di- or tri-alkylamine, mono-, di- or tri-alkanolamine, mono-alkyl-mono-alkanolamine, di-alkyl-mono-alkanolamine, mono-alkyl-di-alkanolamine, etc. A method is mentioned.
- the amount of these halogenated alkyl compounds used is preferably 0.2% by mass or more based on the total mass of the separating material.
- the reaction conditions are preferably 40 to 90 ° C. and 0.5 to 12 hours.
- a strongly basic quaternary ammonium group as an ion exchange group first, a tertiary amino group is introduced, and then the tertiary amino group is reacted with a halogenated alkyl compound such as epichlorohydrin. The method of converting into an ammonium group is mentioned. Further, quaternary ammonium hydrochloride or the like may be reacted with the separation material.
- Examples of the method for introducing a carboxy group that is a weakly acidic group as an ion exchange group include a method in which a monohalogenocarboxylic acid such as monohalogenoacetic acid or monohalogenopropionic acid or a sodium salt thereof is reacted as the halogenated alkyl compound. .
- the amount of the halogenated alkyl compound used is preferably 0.2% by mass or more based on the total mass of the separating material into which the ion exchange group is introduced.
- a glycidyl compound such as epichlorohydrin is reacted with a separating material, and a sulfite or a bisulfite such as sodium sulfite or sodium bisulfite.
- a method of adding a separating material to the saturated aqueous solution are preferably 30 to 90 ° C. and 1 to 10 hours.
- examples of the method for introducing ion exchange groups include a method in which 1,3-propane sultone is reacted with a separation material in an alkaline atmosphere. 1,3-propane sultone is preferably used in an amount of 0.4% by mass or more based on the total mass of the separating material.
- the reaction conditions are preferably 0 to 90 ° C. and 0.5 to 12 hours.
- the moisture absorption of the separation material of the present embodiment is preferably 1 to 30% by mass, more preferably 1 to 20% by mass, and further preferably 1 to 10% by mass.
- the moisture absorption of the separating material is 30% by mass or less, it is possible to suppress a decrease in liquid permeability of the separating material due to the thickness of the coating layer.
- the average pore diameter, mode diameter, specific surface area and porosity in the pore diameter distribution of the separating material or porous polymer particles of the present embodiment are values measured with a mercury intrusion measuring apparatus (Autopore: manufactured by Shimadzu Corporation). Measured as follows. 0.05 g of a sample is added to a standard 5 mL powder cell (stem volume: 0.4 mL) and measured under conditions of an initial pressure of 21 kPa (approximately 3 psia, corresponding to a pore diameter of approximately 60 ⁇ m). Mercury parameters are set to a device default mercury contact angle of 130 degrees and a mercury surface tension of 485 dynes / cm. Each value is calculated by limiting the pore diameter to a range of 0.1 to 3 ⁇ m.
- the separation material of this embodiment is suitable for use in separation of proteins by electrostatic interaction and affinity purification. For example, after adding the separation material of the present embodiment to a mixed solution containing protein, adsorbing only the protein to the separation material by electrostatic interaction, the separation material is filtered from the solution, and the salt concentration If added to a high aqueous solution, the protein adsorbed on the separation material can be easily desorbed and recovered. Moreover, the separation material of this embodiment can also be used in column chromatography.
- FIG. 1 shows an embodiment of a separation column.
- the separation column 10 includes a column 1 and a separation material 2 packed in the column 1.
- a water-soluble substance is preferable.
- proteins such as blood proteins such as serum albumin and immunoglobulin, enzymes present in the living body, protein bioactive substances produced by biotechnology, DNA, biopolymers such as bioactive peptides, etc. Yes, preferably the weight average molecular weight is 2 million or less, more preferably 500,000 or less.
- the separation material of the present embodiment after the coating layer on the porous polymer particles is cross-linked, an ion exchange group, protein A, etc. are introduced into the surface of the separation material, thereby separating natural macromolecules such as proteins.
- Each has the advantage of particles made of a polymer or particles made of a synthetic polymer.
- the porous polymer particles in the separation material of the present embodiment are obtained by the above-described method, they have durability and alkali resistance.
- the separation material of the present embodiment tends to reduce non-specific adsorption of proteins and easily cause protein adsorption / desorption.
- the separation material of the present embodiment tends to have a large adsorption amount (dynamic adsorption amount) of protein or the like under the same flow rate.
- the liquid flow rate represents the liquid flow rate when the separation material of this embodiment is filled in a stainless steel column of ⁇ 7.8 ⁇ 300 mm and the liquid is passed.
- the liquid passing speed is 800 cm / h or more when the column pressure is 0.3 MPa.
- the flow rate of protein solution or the like is generally in the range of 400 cm / h or less.
- the separation rate for normal protein separation is as follows. It can be used at a liquid passing speed of 800 cm / h or more faster than the separating material.
- the average particle size of the separation material of this embodiment is preferably 10 to 300 ⁇ m.
- it is preferably 10 to 100 ⁇ m in order to avoid an extreme increase in column internal pressure.
- the separation material of this embodiment When the separation material of this embodiment is used as a column packing material in column chromatography, it has excellent operability because there is almost no volume change in the column regardless of the properties of the eluate used.
- the 5% compressive deformation elastic modulus of the separating material of the present embodiment can be calculated as follows. Using a micro compression tester (Fisher), the load when particles are compressed to 50 mN with a smooth end face (50 ⁇ m ⁇ 50 ⁇ m) of a quadrangular prism at room temperature (25 ° C.) at a load rate of 1 mN / sec. And measure the compression displacement. From the measured values obtained, the compression modulus (5% K value) when the particles are compressively deformed by 5% can be obtained by the following formula. Further, the load at the point at which the displacement during the measurement changes most greatly is defined as the breaking strength (mN).
- the compression elastic modulus (5% K value) when the separating material is 5% compressively deformed is preferably 100 to 1000 MPa, more preferably 200 to 1000 MPa, and further preferably 250 to 1000 MPa.
- the pore volume (porosity) of the separation material is preferably 30% by volume or more and 70% by volume or less, based on the total volume (including the pore volume) of the separation material, and is 40% by volume or more and 70% by volume or less. It is more preferable.
- the separating material preferably has pores having an average pore diameter of 0.05 to 1 ⁇ m.
- the average pore diameter is preferably 0.2 to 0.5 ⁇ m. If the pore diameter is 0.05 ⁇ m or more, a substance tends to easily enter the pores. If the pore diameter is 1 ⁇ m or less, the specific surface area is sufficient.
- the specific surface area of the separating material is preferably 10 m 2 / g or more, and more preferably 30 m 2 / g or more. From the viewpoint of higher practicality, the specific surface area is more preferably 35 m 2 / g or more, and further preferably 40 m 2 / g or more. When the specific surface area is 10 m 2 / g or more, the adsorption amount of the substance to be separated tends to increase.
- the 5% deformation modulus, mode diameter in the pore size distribution, specific surface area, etc. of the separating material can be adjusted by appropriately selecting the raw material of the porous polymer particles, the porosifying agent, the polymer having a hydroxyl group, and the like. it can.
- this embodiment demonstrated the separation material of the form which introduce
- a separation material can be used for, for example, gel filtration chromatography. That is, the separation column of this embodiment includes a column and the separation material of this embodiment packed in the column.
- Example 1 Synthesis of porous polymer particles> To a 500 mL three-necked flask, add 16 g of 96% pure divinylbenzene (manufactured by Nippon Steel & Sumikin Chemical Co., Ltd., trade name: DVB960), 19.5 g of isoamyl alcohol, 4.5 g of diethylbenzene, and 0.64 g of benzoyl peroxide. An aqueous solution of tricalcium phosphate (TCP) (0.5% by mass) was prepared.
- TCP tricalcium phosphate
- This aqueous solution was emulsified using a microprocess server, and the resulting emulsion was transferred to a flask and stirred for about 8 hours using a stirrer while heating in a water bath at 80 ° C.
- the obtained particles were filtered and washed with acetone.
- TCP was dissolved with an acidic aqueous solution to obtain porous polymer particles 1.
- the specific surface area of the obtained particles and the mode diameter in the pore size distribution were measured by a mercury intrusion method, and the average particle size was measured by a flow type particle size measuring device. The results are shown in Table 1.
- ⁇ Formation and cross-linking of coating layer 4 g of sodium hydroxide and 0.14 g of glycidyl phenyl ether were added to 100 mL of an agarose aqueous solution (2% by mass) and reacted at 70 ° C. for 12 hours to introduce a phenyl group into agarose.
- the obtained modified agarose was reprecipitated with isopropyl alcohol and washed.
- Porous polymer particles 1 are added to a 200 mg / mL modified agarose aqueous solution, and 1 g of porous polymer particles 1 are added to 70 mL of the aqueous solution, and the mixture is stirred at 50 ° C. for 24 hours. Adsorbed.
- the modified agarose was crosslinked as follows. 1 g of porous polymer particles 1 on which modified agarose is adsorbed with respect to 35 mL of the aqueous solution are added to an aqueous solution having ethylene glycol diglycidyl ether and sodium hydroxide concentrations of 0.64 M and 0.4 M, respectively, for 24 hours. Stir at room temperature. Then, after washing
- the amount of agarose adsorbed on the porous polymer particles was measured by weight reduction due to thermal decomposition. 10 mg each of porous polymer particles, modified agarose, and particles (separation material) obtained by the formation and crosslinking of the coating layer were heated from 30 ° C to 900 ° C. Since it was found that the porous polymer particles were thermally decomposed at 500 ° C. and the modified agarose was thermally decomposed at 300 ° C., the modified agarose coating amount of the modified agarose-coated porous polymer particles was estimated from these two data.
- BSA bovine serum albumin
- ⁇ Introduction of ion exchange groups The particles (separation material) obtained by the formation and crosslinking of the coating layer were subjected to centrifugation to remove water, and then poured into a mixture of 5 M sodium hydroxide aqueous solution 20 mL and 5 M diethylaminoethyl hydrochloride aqueous solution 20 mL, The mixture was stirred at 70 ° C. for 12 hours. After completion of the reaction, the mixture was filtered and washed with water to obtain a DEAE-modified separation material having a diethylaminoethyl (DEAE) group as an ion exchange group.
- DEAE diethylaminoethyl
- Example 2 ⁇ Coating layer formation and crosslinking> Synthesis and evaluation were performed in the same manner as in Example 1 except that epichlorohydrin was used instead of ethylene glycol diglycidyl ether.
- Example 3 Synthesis and evaluation were performed in the same manner as in Example 2 except that the amounts of isoamyl alcohol and diethylbenzene were changed to 16 g and 8 g, respectively.
- Example 1 The same evaluation as in Example 1 was performed using the porous polymer particles 1 as they were.
- Comparative Example 3 Commercially available agarose particles (Capto DEAE: GE Healthcare) were used as Comparative Example 3, and the same evaluation as in Example 1 was performed.
- Example 1 the coating amount with the polymer having a hydroxyl group is set within the predetermined range of the present invention. As a result, non-specific adsorption and protein denaturation can be suppressed, and the protein binding capacity and the protein recovery rate were improved as compared with those using agarose particles. Further, Example 2 is different from Example 1 in that the crosslinking agent is changed. In Example 2, nonspecific adsorption and protein denaturation were suppressed as in Example 1, and the ion exchange capacity was further increased and the protein binding capacity was improved. And Example 3 increases the coating amount per unit specific surface area of particle
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Abstract
Description
例えば、多孔質高分子の細孔内に天然高分子ゲル等のゲルを保持した複合体が、ペプチド合成の分野で知られている(特許文献1参照)。特許文献1には、このような複合体を用いることにより、反応性物質の負荷係数を高め、高収率の合成が可能となることが記載されている。 In order to overcome the drawbacks of having a hydrophilic natural polymer cross-linked gel, attempts have been made so far to combine rigid substances that become skeletons.
For example, a complex in which a gel such as a natural polymer gel is held in the pores of a porous polymer is known in the field of peptide synthesis (see Patent Document 1).
[1] モノマ単位として、スチレン及びジビニルベンゼンの少なくとも一方を、モノマ全量基準で90質量%以上含む、多孔質ポリマ粒子と、該多孔質ポリマ粒子の表面の少なくとも一部を被覆する、水酸基を有する高分子を含む被覆層と、を備え、被覆層による被覆量が多孔質ポリマ粒子の単位比表面積当たり1~15mg/m2である分離材。
[2] 上記分離材の表面における酸素元素比率が10~50%である、[1]に記載の分離材。
[3] 上記水酸基を有する高分子が多糖類又はその変性体である、[1]又は[2]に記載の分離材。
[4] 上記多糖類がアガロース、キトサン、アルギン酸及びデキストランから選ばれる少なくとも一種である、[3]に記載の分離材。
[5] 水酸基を有する高分子が架橋されている、[1]~[4]のいずれかに記載の分離材。
[6] 上記多孔質ポリマ粒子における比表面積が10m2/g以上である、[1]~[5]のいずれかに記載の分離材。
[7] 上記多孔質ポリマ粒子の細孔径分布におけるモード径が0.05~1μmである、[1]~[6]のいずれかに記載の分離材。
[8] カラムと、該カラムに充填された上記[1]~[7]のいずれかに記載の分離材とを備える、分離用カラム。 The present invention provides the separating material described in [1] to [6] below.
[1] The polymer unit has at least one of styrene and divinylbenzene at 90% by mass or more based on the total amount of the monomer, and has a hydroxyl group that covers at least a part of the surface of the porous polymer particle. And a coating layer containing a polymer, wherein the coating amount by the coating layer is 1 to 15 mg / m 2 per unit specific surface area of the porous polymer particles.
[2] The separation material according to [1], wherein the oxygen element ratio on the surface of the separation material is 10 to 50%.
[3] The separation material according to [1] or [2], wherein the polymer having a hydroxyl group is a polysaccharide or a modified product thereof.
[4] The separation material according to [3], wherein the polysaccharide is at least one selected from agarose, chitosan, alginic acid, and dextran.
[5] The separating material according to any one of [1] to [4], wherein a polymer having a hydroxyl group is crosslinked.
[6] The separation material according to any one of [1] to [5], wherein the specific surface area of the porous polymer particles is 10 m 2 / g or more.
[7] The separation material according to any one of [1] to [6], wherein the mode diameter in the pore size distribution of the porous polymer particles is 0.05 to 1 μm.
[8] A separation column comprising a column and the separation material according to any one of [1] to [7] packed in the column.
本実施形態の分離材は、多孔質ポリマ粒子と、該多孔質ポリマ粒子の表面の少なくとも一部を被覆する被覆層と、を備える。なお、本明細書中、「多孔質ポリマ粒子の表面」とは、多孔質ポリマ粒子の外側の表面のみでなく、多孔質ポリマ粒子の内部における細孔の表面を含むものとする。 <Separation material>
The separating material of the present embodiment includes porous polymer particles and a coating layer that covers at least part of the surface of the porous polymer particles. In the present specification, the “surface of the porous polymer particle” includes not only the outer surface of the porous polymer particle but also the surface of the pores inside the porous polymer particle.
本実施形態の多孔質ポリマ粒子は、多孔質化剤を含むモノマを硬化させた粒子であり、例えば、従来の懸濁重合、乳化重合等によって合成することができる。モノマ単位としては、スチレン及びジビニルベンゼンの少なくとも一方を、モノマ全量基準で90質量%以上含み、好ましくはジビニルベンゼンをモノマ全量基準で90質量%以上含む。所定量のスチレン又はジビニルベンゼンを含むことにより、耐圧性に優れる傾向にある。
モノマはさらに、以下のようなスチレン及びジビニルベンゼン以外の多官能性モノマ、単官能性モノマ等を含んでいてもよい。 (Porous polymer particles)
The porous polymer particles of the present embodiment are particles obtained by curing a monomer containing a porosifying agent, and can be synthesized by, for example, conventional suspension polymerization or emulsion polymerization. As the monomer unit, at least one of styrene and divinylbenzene contains 90% by mass or more based on the total monomer amount, and preferably 90% by mass or more of divinylbenzene based on the total monomer amount. By containing a predetermined amount of styrene or divinylbenzene, the pressure resistance tends to be excellent.
The monomer may further contain the following polyfunctional monomers other than styrene and divinylbenzene, monofunctional monomers, and the like.
1)粒子を、超音波分散装置を使用して水(界面活性剤等の分散剤を含む)に分散させ、1質量%の多孔質ポリマ粒子を含む分散液を調製する。
2)粒度分布計(シスメックスフロー、シスメックス株式会社製)を用いて、上記分散液中の粒子約1万個の画像により平均粒径及び粒径のC.V.(変動係数)を測定する。 C. of average particle size and particle size of porous polymer particles or separator V. (Coefficient of variation) can be determined by the following measurement method.
1) Disperse the particles in water (including a dispersant such as a surfactant) using an ultrasonic dispersion device to prepare a dispersion liquid containing 1% by mass of porous polymer particles.
2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex Corporation), an average particle size and particle size of C.I. V. (Coefficient of variation) is measured.
本実施形態の被覆層は、所定量(被覆量)の水酸基を有する高分子を含む。所定量の水酸基を有する高分子で多孔質ポリマ粒子を被覆することによりカラム圧の上昇を抑制することができるとともに、タンパク質の非特異吸着を抑制することが可能となる上、分離材のタンパク質吸着量が良好となる傾向にある。さらに、水酸基を有する高分子が架橋されていると、カラム圧の上昇をより抑制することが可能となる。 (Coating layer)
The coating layer of this embodiment contains a polymer having a predetermined amount (coating amount) of hydroxyl groups. By covering the porous polymer particles with a polymer having a predetermined amount of hydroxyl groups, the increase in column pressure can be suppressed, and nonspecific adsorption of proteins can be suppressed, and the protein adsorption of the separation material The amount tends to be good. Furthermore, when the polymer having a hydroxyl group is crosslinked, it is possible to further suppress an increase in column pressure.
水酸基を有する高分子は、1分子中に2個以上の水酸基を有することが好ましく、親水性高分子であることがより好ましい。水酸基を有する高分子としては、例えば、多糖類、ポリビニルアルコール等が挙げられる。多糖類としては、好ましくはアガロース、デキストラン、セルロース、キトサン、アルギン酸等が挙げられる。水酸基を有する高分子としては、例えば重量平均分子量1万~20万程度のものが使用できる。 (Polymer having a hydroxyl group)
The polymer having a hydroxyl group preferably has two or more hydroxyl groups in one molecule, and more preferably a hydrophilic polymer. Examples of the polymer having a hydroxyl group include polysaccharides and polyvinyl alcohol. Preferred examples of the polysaccharide include agarose, dextran, cellulose, chitosan, and alginic acid. As the polymer having a hydroxyl group, for example, a polymer having a weight average molecular weight of about 10,000 to 200,000 can be used.
水酸基を有する高分子を含む被覆層は、例えば、以下に示す方法により形成することができる。
まず、水酸基を有する高分子の溶液を多孔質ポリマ粒子表面に吸着させる。水酸基を有する高分子の溶液の溶媒としては、水酸基を有する高分子を溶解することのできるものであれば、特に限定されないが、水が最も一般的である。溶媒に溶解させる高分子の濃度は、5~20(mg/mL)が好ましい。
この溶液を、多孔質ポリマ粒子に含浸させる。含浸方法は、水酸基を有する高分子の溶液に多孔質ポリマ粒子を加えて一定時間放置する。含浸時間は多孔質体の表面状態によっても変わるが、通常一昼夜含浸すれば高分子濃度が多孔質体の内部で外部濃度と平衡状態となる。その後、水、アルコール等の溶媒で洗浄し、未吸着分の水酸基を有する高分子を除去する。 (Formation method of coating layer)
The coating layer containing a polymer having a hydroxyl group can be formed by, for example, the following method.
First, a polymer solution having a hydroxyl group is adsorbed on the surface of the porous polymer particles. The solvent for the polymer solution having a hydroxyl group is not particularly limited as long as it can dissolve the polymer having a hydroxyl group, but water is the most common. The concentration of the polymer dissolved in the solvent is preferably 5 to 20 (mg / mL).
This solution is impregnated into porous polymer particles. In the impregnation method, porous polymer particles are added to a polymer solution having a hydroxyl group and left for a predetermined time. Although the impregnation time varies depending on the surface state of the porous body, the polymer concentration is in an equilibrium state with the external concentration inside the porous body if it is usually impregnated day and night. Then, it wash | cleans with solvents, such as water and alcohol, and the polymer | macromolecule which has the hydroxyl group which is not adsorbed is removed.
次いで、架橋剤を加えて多孔質ポリマ粒子表面に吸着された水酸基を有する高分子を架橋反応させて、架橋体を形成する。すなわち、分離材の水酸基を有する高分子は架橋されていてもよい。このとき、架橋体は、水酸基を有する3次元架橋網目構造を有する。 (Crosslinking treatment)
Next, a crosslinking agent is added to cause the polymer having a hydroxyl group adsorbed on the surface of the porous polymer particles to undergo a crosslinking reaction to form a crosslinked body. That is, the polymer having a hydroxyl group of the separating material may be cross-linked. At this time, the crosslinked body has a three-dimensional crosslinked network structure having a hydroxyl group.
被覆層を備える分離材は、イオン交換基、リガンド(プロテインA)等を表面上の水酸基等を介して導入することによりイオン交換精製、アフィニティ精製等に使用することができる。イオン交換基の導入方法として、例えば、ハロゲン化アルキル化合物を用いる方法が挙げられる。 (Introduction of ion exchange groups)
The separation material having a coating layer can be used for ion exchange purification, affinity purification, etc. by introducing ion exchange groups, ligands (protein A), etc. via hydroxyl groups on the surface. Examples of the method for introducing an ion exchange group include a method using an alkyl halide compound.
(吸湿後分離材質量-1)g/1g×100=吸湿度(%) The moisture absorption of the separation material of this embodiment is measured by the following method. After 1 g of the dried separation material is left in a constant temperature and humidity test tank (temperature 60 ° C., humidity 90%) for 18 hours, the mass of the separation material is measured again to calculate the moisture absorption from the following equation.
(Separation material mass after moisture absorption -1) g / 1g x 100 = moisture absorption (%)
微小圧縮試験機(Fisher社製)を用いて、室温(25℃)条件にて荷重負荷速度1mN/秒で、四角柱の平滑な端面(50μm×50μm)により粒子を50mNまで圧縮したときの荷重及び圧縮変位を測定する。得られた測定値から、粒子が5%圧縮変形したときの圧縮弾性率(5%K値)を下記式により求めることができる。また、上記測定中の変位量が最も大きく変化する点の荷重を破壊強度(mN)とする。
5%K値(MPa)=(3/21/2)・F・S-3/2・R-1/2
F:架橋ポリマ粒子が40%圧縮変形したときの荷重(mN)
S:架橋ポリマ粒子が40%圧縮変形したときの圧縮変位(mm)
R:架橋ポリマ粒子の半径(mm) The 5% compressive deformation elastic modulus of the separating material of the present embodiment can be calculated as follows.
Using a micro compression tester (Fisher), the load when particles are compressed to 50 mN with a smooth end face (50 μm × 50 μm) of a quadrangular prism at room temperature (25 ° C.) at a load rate of 1 mN / sec. And measure the compression displacement. From the measured values obtained, the compression modulus (5% K value) when the particles are compressively deformed by 5% can be obtained by the following formula. Further, the load at the point at which the displacement during the measurement changes most greatly is defined as the breaking strength (mN).
5% K value (MPa) = (3/2 1/2 ) · F · S −3 / 2 · R −1/2
F: Load (mN) when the crosslinked polymer particles are 40% compressively deformed
S: Compression displacement (mm) when the crosslinked polymer particles are 40% compressed and deformed
R: radius of cross-linked polymer particles (mm)
<多孔質ポリマ粒子の合成>
500mLの三口フラスコに、純度96%のジビニルベンゼン(新日鉄住金化学株式会社製、商品名:DVB960)を16g、イソアミルアルコールを19.5g、ジエチルベンゼンを4.5g、及び過酸化ベンゾイルを0.64g加え、第三リン酸カルシウム(TCP)(0.5質量%)水溶液を調製した。この水溶液をマイクロプロセスサーバーを使用して乳化後、得られた乳化液をフラスコに移し、80℃のウォーターバスで加熱しながら、攪拌機を用いて約8時間攪拌した。得られた粒子をろ過後、アセトンで洗浄を行った。最後に酸性水溶液でTCPを溶解させ、多孔質ポリマ粒子1を得た。得られた粒子の比表面積、及び細孔径分布におけるモード径を水銀圧入法で、平均粒径をフロー型粒径測定装置で測定した。その結果を表1に示す。 (Example 1)
<Synthesis of porous polymer particles>
To a 500 mL three-necked flask, add 16 g of 96% pure divinylbenzene (manufactured by Nippon Steel & Sumikin Chemical Co., Ltd., trade name: DVB960), 19.5 g of isoamyl alcohol, 4.5 g of diethylbenzene, and 0.64 g of benzoyl peroxide. An aqueous solution of tricalcium phosphate (TCP) (0.5% by mass) was prepared. This aqueous solution was emulsified using a microprocess server, and the resulting emulsion was transferred to a flask and stirred for about 8 hours using a stirrer while heating in a water bath at 80 ° C. The obtained particles were filtered and washed with acetone. Finally, TCP was dissolved with an acidic aqueous solution to obtain
アガロース水溶液(2質量%)100mLに水酸化ナトリウム4g、グリシジルフェニルエーテル0.14gを投入し、70℃で12時間反応させ、アガロースにフェニル基を導入した。得られた変性アガロースをイソプロピルアルコールで再沈殿させ、洗浄した。
200mg/mLの変性アガロース水溶液に多孔質ポリマ粒子1を、水溶液70mLに対して多孔質ポリマ粒子1を1gの割合で投入し、50℃で24時間攪拌させ、多孔質ポリマ粒子1に変性アガロースを吸着させた。吸着後、ろ過を行い、熱水で8時間洗浄した。
その結果を表1に示す。
変性アガロースは次のように架橋した。エチレングリコールジグリシジルエーテル及び水酸化ナトリウムの濃度がそれぞれ0.64M、0.4Mである水溶液に、水溶液35mLに対して変性アガロースが吸着した多孔質ポリマ粒子1を1gの割合で投入し、24時間室温にて攪拌した。その後、熱した2質量%のドデシル硫酸ナトリウム水溶液で洗浄後、純水で洗浄し、乾燥させることで分離材を得た。 <Formation and cross-linking of coating layer>
4 g of sodium hydroxide and 0.14 g of glycidyl phenyl ether were added to 100 mL of an agarose aqueous solution (2% by mass) and reacted at 70 ° C. for 12 hours to introduce a phenyl group into agarose. The obtained modified agarose was reprecipitated with isopropyl alcohol and washed.
The results are shown in Table 1.
The modified agarose was crosslinked as follows. 1 g of
多孔質ポリマ粒子へのアガロースの吸着量の測定について、熱分解による重量減少による分析を行った。多孔質ポリマ粒子、変性アガロース、及び上記被覆層の形成及び架橋で得られた粒子(分離材)それぞれ10mgを30℃から900℃まで加温した。多孔質ポリマ粒子は500℃、変性アガロースは300℃で熱分解することが分かったので、この2つのデータから変性アガロース被覆多孔質ポリマ粒子の変性アガロース被覆量を概算した。
具体的には、熱重量分析により、変性アガロース及び多孔質ポリマ粒子の重量減少を測定し、多孔質ポリマ粒子1g当たりの変性アガロース量を算出した。ここで算出した多孔質ポリマ粒子1g当たりの変性アガロース量を比表面積で割ることによって、単位比表面積当たりの被覆量を算出した(下記式(1)参照)。その結果を表1に示す。
単位比表面積あたりの被覆量[mg/m2]=(水酸基を有する高分子の重量減少量[%])/(多孔質ポリマ粒子の重量減少量[%])/(比表面積[m2/g])×1000 …式(1) (Measurement of coating amount on porous polymer particles with modified agarose)
The amount of agarose adsorbed on the porous polymer particles was measured by weight reduction due to thermal decomposition. 10 mg each of porous polymer particles, modified agarose, and particles (separation material) obtained by the formation and crosslinking of the coating layer were heated from 30 ° C to 900 ° C. Since it was found that the porous polymer particles were thermally decomposed at 500 ° C. and the modified agarose was thermally decomposed at 300 ° C., the modified agarose coating amount of the modified agarose-coated porous polymer particles was estimated from these two data.
Specifically, the weight loss of the modified agarose and the porous polymer particles was measured by thermogravimetric analysis, and the amount of the modified agarose per 1 g of the porous polymer particles was calculated. The coating amount per unit specific surface area was calculated by dividing the amount of modified agarose per 1 g of the porous polymer particles calculated here by the specific surface area (see the following formula (1)). The results are shown in Table 1.
Coating amount per unit specific surface area [mg / m 2 ] = (weight reduction amount of polymer having hydroxyl group [%]) / (weight reduction amount of porous polymer particle [%]) / (specific surface area [m 2 / g]) × 1000 (1)
まず、既知濃度のBSA(牛血清アルブミン)水溶液で検量線を作成した(0~0.4%)。上記被覆層の形成及び架橋で得られた粒子(分離材)200mgを20℃の水に12時間浸して膨潤した。その膨潤粒子と水10mLをバイヤル瓶に秤量した。100mLメスフラスコに、BSAを秤量し、リン酸食塩緩衝液(pH=7.1)で溶解し、12mg/mL及び24mg/mLのBSA水溶液を調製した。これらのBSA溶液10mLを上記の粒子溶液に添加し、25℃にて24時間ミックスロータで攪拌した。この溶液の上澄み10mLを10倍に希釈し、吸光度(280nm)を測定し、分離材へのBSA吸着量を非特異吸着量として算出した。その結果を表2に示す。 (Evaluation of nonspecific adsorption ability of protein)
First, a calibration curve was prepared with a known concentration of BSA (bovine serum albumin) aqueous solution (0 to 0.4%). 200 mg of particles (separation material) obtained by the formation and crosslinking of the coating layer were immersed in water at 20 ° C. for 12 hours to swell. The swollen particles and 10 mL of water were weighed into a vial. BSA was weighed into a 100 mL volumetric flask and dissolved in a phosphate buffer solution (pH = 7.1) to prepare 12 mg / mL and 24 mg / mL BSA aqueous solutions. 10 mL of these BSA solutions were added to the particle solution and stirred with a mix rotor at 25 ° C. for 24 hours. 10 mL of the supernatant of this solution was diluted 10 times, the absorbance (280 nm) was measured, and the amount of BSA adsorbed on the separation material was calculated as the amount of non-specific adsorption. The results are shown in Table 2.
上記被覆層の形成及び架橋で得られた粒子(分離材)を、乾燥機で70℃、12時間乾燥させた後、粒子表面の酸素元素比率を下記XPS(X線光電子分光)装置で測定した。その結果を表1に示す。
また、同様の方法で粒子を乾燥させた後、乳鉢で粒子を磨り潰して、粒子内部の酸素元素比率を下記XPS装置で測定した。その結果を表1に示す。
なお、酸素元素比率は、酸素元素に由来するピーク強度を、水素元素以外の元素のピーク強度で割り算することにより、算出した。
XPS装置:アルバック・ファイ社製 PHI 5000 VersaProbeII
X線:単色化AIkα線 (1486.6eV))
分析面積:200μm2
分析深度:50Å (Measurement of oxygen element ratio)
The particles (separation material) obtained by the formation and crosslinking of the coating layer were dried with a dryer at 70 ° C. for 12 hours, and then the oxygen element ratio on the particle surface was measured with the following XPS (X-ray photoelectron spectroscopy) apparatus. . The results are shown in Table 1.
Moreover, after drying particle | grains by the same method, particle | grains were ground with the mortar and the oxygen element ratio inside particle | grains was measured with the following XPS apparatus. The results are shown in Table 1.
The oxygen element ratio was calculated by dividing the peak intensity derived from the oxygen element by the peak intensity of an element other than the hydrogen element.
XPS device: PHI 5000 VersaProbeII manufactured by ULVAC-PHI
X-ray: Monochromatic AIkα ray (1486.6 eV))
Analysis area: 200 μm 2
Analysis depth: 50 mm
上記被覆層の形成及び架橋で得られた粒子(分離材)を、遠心分離により水を除去した後、5M水酸化ナトリウム水溶液20mLと5Mジエチルアミノエチル塩酸塩水溶液20mLとを混合した液に投入し、70℃で12時間攪拌した。反応終了後、ろ過、水洗し、ジエチルアミノエチル(DEAE)基をイオン交換基として有するDEAE変性分離材を得た。 <Introduction of ion exchange groups>
The particles (separation material) obtained by the formation and crosslinking of the coating layer were subjected to centrifugation to remove water, and then poured into a mixture of 5 M sodium hydroxide aqueous solution 20 mL and 5 M diethylaminoethyl hydrochloride aqueous solution 20 mL, The mixture was stirred at 70 ° C. for 12 hours. After completion of the reaction, the mixture was filtered and washed with water to obtain a DEAE-modified separation material having a diethylaminoethyl (DEAE) group as an ion exchange group.
得られたDEAE変性分離材200mgを20℃の水に12時間浸して膨潤した。その膨潤分離材と水10mLをバイヤル瓶に秤量した。100mLメスフラスコに、牛血清アルブミン(BSA)を秤量し、リン酸食塩緩衝液(pH=7.1)で溶解し、1.2mg/mL及び12mg/mLのBSA水溶液を調製した。このBSA溶液10mLを、上記膨潤分離材を秤量したバイヤル瓶に添加し、25℃にて24時間ミックスロータで攪拌した。この溶液の上澄み10mLを10倍に希釈し、吸光度(280nm)を測定し、DEAE変性分離材へのBSA吸着量(結合容量)を計算した。その結果を表2に示す。 (Evaluation of protein binding capacity and recovery rate)
200 mg of the obtained DEAE-modified separating material was immersed in water at 20 ° C. for 12 hours to swell. The swelling separator and 10 mL of water were weighed into a vial. Bovine serum albumin (BSA) was weighed into a 100 mL volumetric flask and dissolved in a phosphate buffer solution (pH = 7.1) to prepare 1.2 mg / mL and 12 mg / mL BSA aqueous solutions. 10 mL of this BSA solution was added to the vial that weighed the swelling separator and stirred with a mix rotor at 25 ° C. for 24 hours. 10 mL of the supernatant of this solution was diluted 10-fold, the absorbance (280 nm) was measured, and the BSA adsorption amount (binding capacity) to the DEAE-modified separation material was calculated. The results are shown in Table 2.
タンパク質回収率=(タンパク質脱着後吸光度―タンパク質脱着前吸光度)/(タンパク質吸着後吸光度―タンパク質吸着前吸光度) …式(2) NaCl 0.9g was added to this vial and stirred at 25 ° C for 12 hours with a mix rotor. The supernatant (5 mL) of this solution was diluted 10-fold, the absorbance (280 nm) was measured, and the BSA recovery rate by the separating material was calculated as shown in Equation (2). The results are shown in Table 2.
Protein recovery rate = (absorbance after protein desorption−absorbance before protein desorption) / (absorbance after protein adsorption−absorbance before protein adsorption) (2)
12時間以上水で膨潤させたDEAE変性分離材0.2~0.3gを定量し、ビーカに移し、0.1Nの水酸化ナトリウム溶液20mLを入れ、攪拌した。その後、吸引ろ過を行い、フィルタ上の粒子を、洗浄液が中性になるまで洗浄した。その後、ビーカに移し、0.1N塩酸水溶液20mLを添加し、室温で1時間攪拌した。その後、吸引ろ過を行い、フィルタ上の粒子を、洗浄液が中性になるまで洗浄した。この洗浄液を、0.1N水酸化ナトリウム水溶液で、自動電位差滴定装置を使用して、滴定を行い、イオン交換容量を測定した。その結果を表2に示す。 (Quantification method of ion exchange groups)
DEAE-modified separation material 0.2 to 0.3 g swollen with water for 12 hours or more was quantified, transferred to a beaker, and 20 mL of 0.1 N sodium hydroxide solution was added and stirred. Thereafter, suction filtration was performed, and the particles on the filter were washed until the washing solution became neutral. Then, it moved to the beaker, 20 mL of 0.1N hydrochloric acid aqueous solution was added, and it stirred at room temperature for 1 hour. Thereafter, suction filtration was performed, and the particles on the filter were washed until the washing solution became neutral. This washing solution was titrated with a 0.1N aqueous sodium hydroxide solution using an automatic potentiometric titrator, and the ion exchange capacity was measured. The results are shown in Table 2.
得られたDEAE変性分離材をφ7.8×300mmのステンレスカラムに濃度30質量%のスラリー(溶媒:メタノール)として15分かけて充填した。その後、カラムに流速を変えながら水を流し、線流速とカラム圧の関係を測定し、0.3MPa時の線流速(通液速度)を測定した。その結果を表1に示す。 (Column characteristic evaluation)
The obtained DEAE-modified separation material was packed in a stainless steel column of φ7.8 × 300 mm as a slurry (solvent: methanol) having a concentration of 30% by mass over 15 minutes. Thereafter, water was allowed to flow through the column while changing the flow rate, the relationship between the linear flow rate and the column pressure was measured, and the linear flow rate (liquid flow rate) at 0.3 MPa was measured. The results are shown in Table 1.
<被覆層の形成及び架橋>において、エチレングリコールジグリシジルエーテルに代えて、エピクロロヒドリンを使用した以外は実施例1と同様にして、合成及び評価を行った。 (Example 2)
<Coating layer formation and crosslinking> Synthesis and evaluation were performed in the same manner as in Example 1 except that epichlorohydrin was used instead of ethylene glycol diglycidyl ether.
<多孔質ポリマ粒子の合成>において、イソアミルアルコール及びジエチルベンゼンの使用量を、それぞれ16g、8gに変更した以外は実施例2と同様にして、合成及び評価を行った。 (Example 3)
<Synthesis of porous polymer particles> Synthesis and evaluation were performed in the same manner as in Example 2 except that the amounts of isoamyl alcohol and diethylbenzene were changed to 16 g and 8 g, respectively.
多孔質ポリマ粒子1をそのまま用いて、実施例1と同様の評価を行った。 (Comparative Example 1)
The same evaluation as in Example 1 was performed using the
<被覆層の形成及び架橋>において、変性アガロース水溶液の濃度を20mg/mLに変更した以外は実施例1と同様にして、合成及び評価を行った。 (Comparative Example 2)
<Coating layer formation and cross-linking> Synthesis and evaluation were performed in the same manner as in Example 1 except that the concentration of the modified agarose aqueous solution was changed to 20 mg / mL.
市販のアガロース粒子(Capto DEAE:GEヘルスケア)を比較例3として使用し、実施例1と同様の評価を行った。 (Comparative Example 3)
Commercially available agarose particles (Capto DEAE: GE Healthcare) were used as Comparative Example 3, and the same evaluation as in Example 1 was performed.
Claims (8)
- モノマ単位として、スチレン及びジビニルベンゼンの少なくとも一方を、モノマ全量基準で90質量%以上含む、多孔質ポリマ粒子と、
該多孔質ポリマ粒子の表面の少なくとも一部を被覆する、水酸基を有する高分子を含む被覆層と、を備え、
前記被覆層による被覆量が、前記多孔質ポリマ粒子の単位比表面積当たり1~15mg/m2である分離材。 Porous polymer particles containing at least one of styrene and divinylbenzene as a monomer unit in an amount of 90% by mass or more based on the total amount of monomers;
A coating layer containing a polymer having a hydroxyl group, covering at least a part of the surface of the porous polymer particles,
A separation material in which the coating amount by the coating layer is 1 to 15 mg / m 2 per unit specific surface area of the porous polymer particles. - 前記分離材の表面における酸素元素比率が10~50%である、請求項1に記載の分離材。 The separation material according to claim 1, wherein the oxygen element ratio on the surface of the separation material is 10 to 50%.
- 前記水酸基を有する高分子が多糖類又はその変性体である、請求項1又は2に記載の分離材。 The separation material according to claim 1 or 2, wherein the polymer having a hydroxyl group is a polysaccharide or a modified product thereof.
- 前記多糖類がアガロース、キトサン、アルギン酸及びデキストランから選ばれる少なくとも一種である、請求項3に記載の分離材。 The separation material according to claim 3, wherein the polysaccharide is at least one selected from agarose, chitosan, alginic acid, and dextran.
- 前記水酸基を有する高分子が架橋されている、請求項1~4のいずれか一項に記載の分離材。 The separation material according to any one of claims 1 to 4, wherein the polymer having a hydroxyl group is crosslinked.
- 前記多孔質ポリマ粒子における比表面積が10m2/g以上である、請求項1~5のいずれか一項に記載の分離材。 The separation material according to any one of claims 1 to 5, wherein a specific surface area of the porous polymer particles is 10 m 2 / g or more.
- 前記多孔質ポリマ粒子の細孔径分布におけるモード径が0.05~1μmである、請求項1~6のいずれか一項に記載の分離材。 The separation material according to any one of claims 1 to 6, wherein a mode diameter in a pore size distribution of the porous polymer particles is 0.05 to 1 µm.
- カラムと、該カラムに充填された請求項1~7のいずれか一項に記載の分離材とを備える、分離用カラム。
A separation column comprising a column and the separation material according to any one of claims 1 to 7 packed in the column.
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